Diverse Secondary Metabolites from the Coral-Derived Fungus Aspergillus hiratsukae SCSIO 5Bn1003

Three new metabolites, including a cyclic tetrapeptide asperhiratide (1), an ecdysteroid derivative asperhiratine (2), and a sesquiterpene lactone asperhiratone (3), were isolated and identified from the soft coral-derived fungus Aspergillus hiratsukae SCSIO 5Bn1003, together with 10 known compounds. Their structures were elucidated via spectroscopic analysis, X-ray diffraction analysis, and electronic circular dichroism calculations. In addition, the absolute configuration of 1 was determined by Marfey’s technique and an analysis of the acid hydrolysates using a chiral phase HPLC column. Among all the compounds, 6 and 8 showed medium cytotoxic activities against four tumor cell lines (SF-268, HepG-2, MCF-7, and A549), with IC50 values ranging from 31.03 ± 3.04 to 50.25 ± 0.54 µM. Meanwhile, they strongly inhibited α-glucosidase activities, with IC50 values of 35.73 ± 3.94 and 22.00 ± 2.45 µM, which were close to and even stronger than the positive control acarbose (IC50 = 32.92 ± 1.03 µM). Compounds 6–8 showed significant antibacterial activities against Bacillus subtilis, with MIC values of 10.26 ± 0.76 µM, 17.00 ± 1.25 µM, and 5.30 ± 0.29 µM, respectively. Compounds 9 and 12 exhibited potent radical scavenging activities against DPPH, with IC50 values of 12.23 ± 0.78 µM and 7.38 ± 1.16 µM. In addition, asperhiratide (1) was evaluated for anti-angiogenic activities in the in vivo zebrafish model, which showed a weak inhibitory effect on intersegmental vessel (ISV) formation.


Introduction
The coral reef ecosystem is one of the oldest ecological types with the highest species diversity and productivity on the Earth. It harbors abundant microbial communities, such as fungi, bacteria, actinomycetes, and cyanobacteria [1]. The complexity and extreme conditions marine organisms faced in their niche for survival provide them with a unique potential to produce a large range of compounds [2]. Among all coral microorganisms, fungi have emerged as an alternative source of promising bioactive agents, including alkaloids, terpenoids, polyketides, lipids, proteins, glycosides, and so on. At this point, more than 326 new structural metabolites have been reported from coral fungi [3,4].
Fungal species belonging to the genus Aspergillus have been widely studied. In the marine environment, several Aspergillus species have proved their potential to produce a plethora of secondary metabolites, which display a variety of pharmacological activities, such as antimicrobial, cytotoxic, anti-inflammatory, and antioxidant activity. From 2015 until December 2020, about 361 secondary metabolites were identified from different marine Aspergillus species [5]. However, it should be emphasized that there are still many rare and unstudied Aspergillus species, such as Aspergillus hiratsukae. In our continuing search for bioactive compounds from fungi collected in the South China Sea [6][7][8], we isolated the fungal strain Aspergillus hiratsukae SCSIO 5Bn 1 003 from an unidentified coral sample. Its crude extract exhibited various antibacterial activities in the pre-active screening, including Bacillus subtilis, Staphylococcus aureus, Bacillus thuringiensis, Vibrio alginolyticus XSBZ14, Multi-resistant Pseudomonas aeruginosa, and Micrococcus lutea. A further chemical investigation led to the isolation of three new metabolites, asperhiratide (1), asperhiratine (2), and asperhiratone (3) (Figure 1), as well as 10 known compounds. Herein, we report these compounds' isolation, structure elucidation, and bioactivity evaluation.

Bioactivity Evaluation
All compounds were evaluated for their in vitro cytotoxicity against four human cancer lines, SF-268 (human glioblastoma carcinoma), MCF-7 (breast cancer), HepG-2 (liver cancer), and A549 (lung cancer), as well as antioxidative activity, α-glucosidase inhibitory activity, and antimicrobial activity against the bacteria Staphylococcus aureus and Bacillus subtilis (Tables S1-S3). Compounds 6 and 8 showed moderate cytotoxic activities against all four tumor cell lines, with IC 50 values ranging from 31.03 ± 3.04 to 50.25 ± 0.54 µM (Table 2). Meanwhile, they exhibited significant inhibitory activity against α-glucosidase, with IC 50 values of 35.73 ± 3.94 and 22.00 ± 2.45 µM, which are even better or comparable to that of the positive control acarbose (IC 50 = 32.92 ± 1.03 µM) (  (Table 3). In addition, Compound 1 was also evaluated for anti-angiogenic activities in the in vivo zebrafish model, which showed a weak inhibitory effect on intersegmental vessel (ISV) formation ( Figure 5).

General
NMR spectra were recorded on an AVANCE III HD-700 spectrometer (Bruker, Billerica, MA, USA) with TMS as the internal standard. HR-ESI-MS spectra data were gathered on a MaXis quadrupole time-of-flight mass spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany). Crystallographic data were collected on a Rigaku XtaLAB AFC12 single-crystal diffractometer (Rigaku, Kyoto, Japan) using Cu Kα radiation. Optical rotations were measured with an MCP500 automatic polarimeter (AntonPaar, Graz, Austria) with MeCN as the solvent. UV spectra were recorded on a UV-2600 spectrometer (Shimadzu, Tokyo, Japan). Circular dichroism spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics, Ltd., Surrey, Leatherhead, UK) with the same concentration of UV measurement (Pathlength 10 mm, Applied Photophysics, Ltd., Surrey, Leatherhead, UK). HPLC purification was performed on an Agilent 1260 HPLC equipped with a DAD detector, using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 µM, 3 mL/min). Silica gel (200-300 and 300-400 mesh) and Sephadex LH-20 for column chromatography (CC) were purchased from Qingdao Marine Chemical Group Co. (Qingdao, China) and GE Healthcare (Uppsala, Sweden), respectively. All solvents used in CC and HPLC were of analytical grade (Tianjin Damao Chemical Plant, Tianjin, China) and chromatographic grade (MERDA, Beijing, China), respectively.

Fungal Materials
The strain Aspergillus hiratsukae SCSIO 5Bn 1 003 was isolated from a coral sample collected from the South China Sea in September 2019. The internal transcribed spacer (ITS1-5.8S-ITS2) regions of the strain were amplified with the universal ITS primers, ITS1 (5 -CTTGGTCATTTAGAGGAAGTAA-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ), using polymerase chain reaction (PCR). The amplified products were submitted for sequencing and aligned with the sequences in the GenBank by Basic Local Alignment Search Tool (BLAST) programs to find out the sequence homology with closely related organisms. When the top three matching BLAST hits were from the same species and were ≥98% similar to the query sequence, this species name was assigned to the selected isolate. Finally, the strain was identified as A. hiratsukae under the accession number KY806121.1. The working strain was deposited with the Research Network for Applied Microbiology (RNAM) Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences.

Fermentation, Extraction, and Purification
The strain was reactivated with a PDA plate, transferred to a 500 mL flask containing 150 mL of fermentation medium (potato extract 6.0 g, glucose 20.0 g, artificial sea salt 30.0 g, distilled water 1.0 L), and cultured at 28 • C, 180 r/min shaker, for 3 days to obtain a seed solution. Then, 5 mL of the seed solution was inoculated into 1000 mL flasks containing 300 mL of fermentation medium, and cultured at 28 • C static for 30 days to obtain 50 L of fermentation broth. The fermentation broth was filtered through a sieve and divided into the filtrate and mycelia. The filtrate was concentrated under reduced pressure, and was extracted with an equal volume of ethyl acetate three times to obtain an ethyl acetate extract of the fermentation broth. The mycelia were extracted with an 80% aqueous acetone solution three times, concentrated under reduced pressure until acetone-free, and extracted with an equal volume of ethyl acetate three times to obtain an ethyl acetate extract of mycelia. The ethyl acetate extracts were combined and concentrated to dryness under reduced pressure to give 25.8 g of crude product.

X-ray Crystal Structure Analysis
Crystallographic data for 1 and 2 were collected on a Rigaku XtaLAB AFC12 singlecrystal diffractometer using Cu Kα radiation. The crystal was kept at 99.9(5) K during data collection. Using Olex2, the structure was solved with the ShelXT structure solution program using intrinsic phasing, and was refined with the ShelXL refinement package using least-squares minimization. Crystallographic data for 1 and 2 have been deposited in the Cambridge Crystallographic Data Centre database (deposition numbers 2,094,392 and 2004886). Copies of the data can be obtained free of charge from the CCDC at www.ccdc. cam.ac.uk (accessed on 9 July 2021). Crystal

Cytotoxic and Antioxidative Assays
The details of the experimental procedures for cytotoxic and antioxidative bioassays were described previously in our former paper [22].

Antibacterial Assay
All the compounds were tested for antibacterial activity against Staphylococcus aureus and Bacillus subtilis using the Mueller-Hinton broth microdilution method [23]. Ciprofloxacin was used as a positive control.

α-Glucosidase Inhibitory Assay
The anti-α-glucosidase activity of the active compounds was evaluated by measuring the increment of absorbance due to the hydrolysis of PNP-G by α-glucosidase at the wavelength of 410 nm with a microplate reader, according to the referenced described method [24].

Zebrafish Assays for In Vivo Anti-Angiogenic Activity
FLI-1: EGFP transgenic zebrafish larvae, purchased from the Molecular Toxicology Research Center of Oregon State University (Covallis, OR, USA), exhibited green fluorescence under fluorescence spectroscopy (Fluorescence Inverted Microscope, Nikon TE2000-U, Tokyo, Japan). The maintenance of zebrafish and the collection of embryos followed the method in the literature [25]. One day after fertilization, the embryos were dechorionated with tweezers in a Petri dish coated with 1% agarose and distributed into small Petri dishes (35 × 10 mm) with 3 mL of assay solution, depending on the drug treatment. The embryos receiving a mixture of DMSO (0.1%) were used as the vehicle-treated control and were equivalent to a "no treatment" group. All the experiments were repeated at least three times, with 20 embryos per group.

Conclusions
In summary, the chemical investigation of a soft coral-derived fungus Aspergillus hiratsukae SCSIO 5Bn 1 003, collected from the South China Sea, led to the discovery of 13 compounds with diverse chemical structures, comprising cyclic peptide, ecdysteroid, sesquiterpene lactone, triterpene, butyrolactones, benzaldehyde, bisphthalate, and coumarin. The results demonstrated the enormous biosynthetic potential of this fungus strain. Asperhiratide (1) contains a rare Gly residue and showed a weak inhibitory effect on intersegmental vessel (ISV) formation. Asperhiratine (2) is a typical phytoecdysteroid which possesses a cholest-7-en-6-one carbon skeleton, a γ-lactone, and a benzoate group. Most phytoecdysteroids are present in a large number of plant families within the angiosperms, gymnosperms, ferns, and terrestrial fungi, but are very rare in marine fungi. Asperhiratone (3) represents an unprecedented sesquiterpene lactone, which possesses a new 6/4-fused carbocyclic core skeleton containing a lactone ring system. In addition, compounds 6-9 and 12 exhibited various activities, such as antimicrobial, cytotoxic, α-glucosidase inhibitory, and antioxidant activity. The above findings demonstrated the huge potentials of coral-associated fungi for the discovery of structurally novel and pharmacologically active natural products.